Fractionated radiotherapy and chemotherapy with an oxygen therapeutic

ABSTRACT

A fluorocarbon emulsion in water for use in fractionated radiotherapy and chemotherapy, wherein said fluorocarbon comprises between 4 and 8 carbon atoms.

FIELD OF THE INVENTION

The invention is directed to use of an oxygen therapeutic in combinationwith repeated administrations of chemotherapy medications and/orradiation therapy.

BACKGROUND OF THE INVENTION

A radiosensitizer is a drug that makes tumor cells more sensitive toradiation therapy. One of the major limitations of radiotherapy is thatthe cells of solid tumors become deficient in oxygen. Solid tumors canoutgrow their blood supply, causing a low-oxygen state known as hypoxia.Oxygen is a potent radiosensitizer, increasing the effectiveness of agiven dose of radiation by forming DNA-damaging free radicals. Tumorcells in a hypoxic environment may be 3 times more resistant toradiation damage than those in a normal oxygen environment.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood from a reading of the followingdetailed description taken in conjunction with the drawings in whichlike reference designators are used to designate like elements, and inwhich:

FIG. 1A graphically shows Oxygen levels in 9 Hs-766T pancreatic cancertumor xenografts in mice. The different traces (lines) in all represent9 tumors in 9 different mice. Each mouse was given a single dose ofNVX-108: 0.3 mL/kg (blue traces), 0.45 mL/kg (green traces) or 0.6 mL/kg(red traces);

FIG. 1B graphically shows average and standard error oxygen levels inHs-766T pancreatic cancer tumor xenografts in mice at the 3 differentdoses of NVX-108: 0.3 mL/kg (blue traces, n=2), 0.45 mL/kg (greentraces, n=3) or 0.6 mL/kg (red traces, n=4);

FIG. 2A graphically shows a comparison of percent tumor growth among 3groups of mice; and

FIG. 2B is an expanded view of the two treated groups of FIG. 2A.

FIG. 3 shows MRI scan images.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This invention is described in preferred embodiments in the followingdescription with reference to the Figures, in which like numbersrepresent the same or similar elements. Reference throughout thisspecification to “one embodiment,” “an embodiment.” or similar languagemeans that a particular feature, structure, or characteristic describedin connection with the embodiment is included in at least one embodimentof the present invention. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment.” and similar language throughout thisspecification may, but do not necessarily, all refer to the sameembodiment.

The described features, structures, or characteristics of the inventionmay be combined in any suitable manner in one or more embodiments. Inthe following description, numerous specific details are recited toprovide a thorough understanding of embodiments of the invention. Oneskilled in the relevant art will recognize, however, that the inventionmay be practiced without one or more of the specific details, or withother methods, components, materials, and so forth. In other instances,well-known structures, materials, or operations are not shown ordescribed in detail to avoid obscuring aspects of the invention.

Dodecafluoropentane emulsion (DDFPe) was previously tested as asensitizer for radiotherapy with a single fraction of radiotherapy.Tumors (xenografts) were irradiated, the tumors removed from theanimals, the cells disaggregated and tested for viability. Neither tumorgrowth nor survival was assessed and tumor pO₂ was not directlyassessed. Nor were the effects of administration of DDFPe withchemotherapy assessed. Multi-dose administration of DDFPe withfractionated radiotherapy was not assessed.

In certain embodiments, the animal to be treated is a mammal. In certainembodiments, the animal to be treated is a human. In certainembodiments, doses range from about 0.01 cc/kg to about 1.0 cc/kg (2%w/vol DDFPe). In certain embodiments, the doses range from about 0.05cc/kg to 0.3 cc/kg administered by infusion for up to 30 minutes or asingle bolus. As one skilled in the art would recognize, if theconcentration of DDFP in the emulsion is increased, e.g. to 5% or 10% byweight, the volume administered will generally be decreased accordingly.Preferably the subject is breathing oxygen or a mixture of oxygen andCO₂, e.g. carbogen, between 95% oxygen with 5% CO₂ to 98% oxygen with 2%CO₂. Applicant has discovered that use of carbogen and oxygen arecomparable, but carbogen is problematic. It has to be ordered speciallywhereas oxygen is available everywhere.

DDFPe is administered IV prior to each fraction of radiation therapy. Incertain embodiments, the DDFPe is administered as a product currently inclinical development under the name NVX-108.

In the prior art, relatively high molecular weight fluorocarbons havebeen studies as radiosensitizers. Materials that have been studied asradiosensitizers include F-1,3-dimethyladamantane,F-trimethylbicyclo[3.3.1]nonane, F-tributylamine (FC-43,″ 3M Company),perfluorodecalin and perfluorooctylbromide.

The inventor has discovered that lower molecular weight fluorocarbons(FC), most particularly with boiling points from about −4 degreescentigrade to about 100 degrees centigrade are far more effective thanthe higher molecular weight, higher boiling point FCs. More preferablythe boiling point of the FC is from about 20 to about 80 degrees C. andstill more preferably from about 28 degrees C. to about 60 degrees C.FCs useful in this invention include perfluorobutane, perfluoropentane,perfluorohexane, perfluoroheptane and perfluorooctane. Most preferredare perfluoropentane and perfluorohexane with the most preferred beingperfluoropentane.

Most preferably the FC is prepared as an emulsion by high pressure,temperature controlled homogenization. A variety of surfactants may beused to prepare the emulsions. The preferred surfactants arephospholipids and a preferred composition of phospholipids includesdioleoylphosphatidylcholine (DOPC) anddioleoylphosphatidylethanolamine-PEG-5,000 (DOPE-PEG 5 k). Anotherpreferred mixture of phospholipids includedipalmitoylphosphatidylcholine (DPPC), anddipalmitoylphosphatidylethanolamine-PEG-5.000 (DPPE-PEG 5 k). Apreferred ratio of lipids is 92 mole percent DPPC with 8 mole percentDPPE-PEG. The same ratio of lipids is also preferred for the unsaturatedphosphatidyl moieties. Other lipids such as cholesterol, phosphatidicacid, stearic acid, palmitic acid, oleic acid andphosphatidylethanolamine may be mixed with the above mentioned lipids.Other useful surfactants include fluorosurfactants such as PEG Telomer Band CAPSTONE (DuPont). Mixtures of phospholipids and thefluorosurfactants may be used. Other surfactants includepolyoxyethylene-polyoxypropylene copolymer surfactant e.g. “Pluronic”F-68.

Preferably a viscogen is included in the formulation to increase theviscosity in the product to decrease settling of the nano-emulsion.Viscogens include sucrose, carboxymethylcellulose, trehalose, starch,Hextend®, xanthan gum, propylene glycol, glycerol and polyethyleneglycol ranging in molecular weight from about 400 to 8,000 MW.Preferably the formulation also includes a buffer such as sodiumphosphate to stabilize the pH near neutrality, i.e. pH=7.0.

Because of the greater efficacy of the present invention using the lowerboiling point FCs much lower doses can be administered to effectivelyreverse radiation resistance. For example, NVX-108 only has 2% w/volDDFP. Prior materials had >10% w/vol FC. For this invention thepreferred weight range is from about 1% to 5% w/vol FC with 2% w/vol FCmost preferred, e.g. 2% w/vol DDFP or perfluorohexane.

Experiments in animals bearing tumor xenografts showed that the effectsof NVX-108 on tumor pO₂ were comparable on animals breathing carbogenand oxygen. But the effect on tumor pO₂ was less in animals breathingroom air. Therefore for the purpose of this invention the subject maybreathe either carbogen or supplemental oxygen during and/or afteradministration of the emulsion and during radiotherapy or administrationof chemotherapy.

The material may be administered concomitantly with radiotherapy orprior to radiotherapy, e.g. up to about 120 minutes prior toradiotherapy. Optionally, in addition to radiotherapy, or alone,chemotherapy is administered concomitantly with DDFPe. A variety ofanti-neoplastic agents may be employed in the invention including butnot limited to alkylating agents, antimetabolites, anthracyclines,topoisomerase inhibitors, mitotic inhibitors, corticosteroids,miscellaneous chemotherapy drugs, targeted therapies, hormone therapyand immunotherapy.

NVX-108 comprises a formulation having the components recited in Table1.

TABLE 1 INGREDIENT SPECIFICATION PURPOSE CONC (MG/ML)DODECAFLUOROPENTANE MEDICAL GRADE ACTIVE 20 SUCROSE MEDICAL GRADEEXCIPIENT 300 PEG TELOMER B PURIFIED CHEMICAL EXCIPIENT 3 GRADE WATERFOR INJECTION USP SOLVENT Q.S. TO 1 ML NITROGEN MEDICAL GRADE HEAD SPACEQ.S. AIR FLUSH SODIUM PHOSPHATE USP BUFFER 0.01M HYDROCHLORIC ACID USPEXCIPIENT Q.S.

As a general matter, Applicant's fluorocarbon emulsion does not compriseany amidoamine oxide compounds. More specifically, Applicant'sfluorocarbon emulsion does not comprise any fluorinated amidoamine oxidecompounds.

Alkylating agents include but are not limited to nitrogen mustards: suchas mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide(Cytoxan®), ifosfamide, and melphalan; nitrosoureas: which includestreptozocin, carmustine (BCNU), and lomustine; alkyl sulfonates:busulfan; triazines: dacarbazine (DTIC) and temozolomide (TEMODAR);ethylenimines: thiotepa and altretamine (hexamethylmelamine). Theplatinum drugs (cisplatin, carboplatin, and oxalaplatin) are sometimesgrouped with alkylating agents because they kill cells in a similar way.

Examples of antimetabolites include: 5-fluorouracil (5-FU),6-mercaptopurine (6-MP), capecitabine (XELODA), cladribine, clofarabine,cytarabine (ARA-C), floxuridine, fludarabine, gemcitabine (GEMZAR),hydroxyurea, methotrexate, pemetrexed (ALIMTA), pentostatin andthioguanine. Anthracyclines include: daunorubicin, doxorubicin(ADRIAMYCIN) and epirubicin. Idarubicin anti-tumor antibiotics that arenot anthracyclines include: actinomycin-bleomycin, and mitomycin-C.Mitoxantrone is an anti-tumor antibiotic that is similar to doxorubicinin many ways. Examples of topoisomerase I inhibitors include topotecanand irinotecan (CPT-11). Examples of topoisomerase II inhibitors includeetoposide (VP-16) and teniposide. Mitoxantrone also inhibitstopoisomerase II. Examples of mitotic inhibitors include: taxanes:paclitaxel (TAXOI) and docetaxel (TAXOTERE). Epothilones includeixabepilone (IXEMPRA). Vinca alkaloids include vinblastine (VELBAN),vincristine (ONCOVIN), and vinorelbine (NAVELBINE) and estramustine(EMCYT). Examples of corticosteroids include prednisone,methylprednisolone (SOLUMEDROL), and dexamethasone (DECADRON). Examplesof targeted therapies include imatinib (GLEEVEC), gefitinib (IRESSA),sunitinib (SUTENT) and bortezomib (VELCADE).

Examples of differentiating agents include the retinoids, tretinoin(ATRA or ATRALIN) and bexarotene (TARGRETIN), as well as arsenictrioxide (ARSENOX). Examples of hormone therapy agents include theanti-estrogens: fulvestrant (FASLODEX), tamoxifen, and torcmifene(FARESTON). Aromatase inhibitors include: anastrozole (ARIMIDEX),exemestane (AROMASIN), and letrozole (FEMARA). Progestins includemegestrol acetate (MEGACE) and estrogens. Anti-androgens includebicalutamide (CASODEX), flutamidc (EULEXIN), and nilutamde (NILANDRON).Gonadotropin-releasing hormone (GnRH), also known as luteinizinghormone-releasing hormone (LHRH) agonists or analogs include leuprolide(LUPRON) and goserelin (ZOLADEX).

Types of immunotherapies and some examples include: monoclonal antibodytherapy (passive immunotherapies), such as rituximab (RITUXAN) andalemtuzutnab (CAMPATH). Non-specific immunotherapies and adjuvants(other substances or cells that boost the immune response) include BCG,interleukin-2 (IL-2), and interferon-alfa. Immunomodulating drugs, forinstance, thalidomide and lenalidomide (REVLIMID). Cancer vaccines(active specific immunotherapies such as PROVENGE vaccine for advancedprostate cancer may be used with DDFPe and other vaccines currentlyunder development. Administration of the FC increases the activity ofthe immunotherapy, for example Yervoy® (Ipilimumab) as the immune cellsare more active in an oxidative environment achieved through tumorre-oxygenation. Other immune modulating drugs that can be used with theFC emulsions include Inhibitors of PD-L1 expression. Monoclonalantibodies are particularly useful with the invention. The antibodiescan be used as vaccines to trigger an immune response to reject thecancer. Non-specific stimulators of the immune system can be used in theinvention. Examples include cytokines such as interleukins andinterferons such as Interferon-alpha and Interleukin-2: Antibodiesuseful in this invention include Alemtuzumab, Bevacizumab, Brentuximabvedotin, Cetuximab, Gemtuzumab ozogamicin, Ibritumomab tiuxetan,lpilimumab, Nivolumab, Ofatumumab, Panitumumab, Rituximab, Tositumomaband Trastuzumab. The invention can be used with Adoptive T-cell therapyAnti-CD47 antibodies, Anti-GD2 antibodies, Immune checkpoint blockadeand EGF receptor antibodies. Administration of the emulsion can be usedto increase the oxygen in the tumor tissue so that immune mechanismsaccelerated of oxidative stress through increasing oxygen and making theimmune system more efficient but also by changing gene expression. Bydecreasing expression of hypoxia related genes the oxygen therapeuticconverts the aggression hypoxia mediated phenotype to a less aggressivephenotype that is more easily defeated by the immune system. In certainembodiments, Applicant's invention includes a method to alter geneexpression by administering to a patient in need thereof, atherapeutically effective dosage of an oxygen therapeutic, such as andwithout limitation, NVX-108.

Administration of DDFPe, preferably while breathing supplemental oxygen,or carbogen, increases tumor oxygenation, enabling the chemotherapeuticdrugs to work more effectively. Synergy is furthermore attained bysimultaneous radiotherapy.

The following examples are presented to further illustrate to personsskilled in the art how to make and use the invention. These examples arenot intended as a limitation, however, upon the scope of the invention.

Example 1 Preparation of Dodecafluoropentane Emulsion

A 30% sucrose solution was prepared by dissolving appropriate amount ofUSP grade sucrose in water for injection at room temperature followed bysodium dihydrogen phosphate to buffer the system at a pH of 7.0. In asecond vessel a suspension of DDFP (dodecafluoropentane) in Peg TelomerB in the ratio of DDFP:PEG Telomer B: 7:1 (w:w), was prepared asfollows. PEG Telomer B was dispersed in water for injection by stirringin a jacketed vessel cooled to 4° C. Pre-cooled (4° C.) DDFP was addedto the stirred PEG Telomer B and allowed to stir until a uniformly milkysuspension was achieved. This suspension was homogenized under highpressure in an Avestin model C50 homogenizer for up to 18 minuteskeeping the temperature below 7° C.). The emulsion was transferred viathe homogenizer under low pressure to a vessel containing 30% sucrosesolution in water; the resulting solution was stirred for up to 20minutes, and then transferred through the homogenizer under low pressureto a second vessel. This solution was then transferred through a 0.2micron filter into a third vessel. The product was dispensed to vials,which were capped and crimped. These operations were carried out at <8°C. in cold jacketed vessels due to the volatility of the activeingredient (DDFP). Compensation for losses during processing wereaccounted for by the use of an overage of the active component. Productfill volume was also tightly controlled to produce vials to meet releaseand shelf-life specifications. The resulting product comprised 2% w/volDDPE. Particle sizing by Nycomps showed mean particle size of about 250nm.

Example 2

An experiment was performed in Hs-766t pancreatic cancer xenograftsimplanted in mice. The Hs-766T (pancreatic; ATCC Cat #HTB-134) cell lineutilized for this study was obtained from the American Type CultureCollection (ATCC, Manassas, Va.) and was handled, stored and managed bythe University of Arizona Experimental Mouse Shared Services (EMSS,University of Arizona). Cells were grown in DMEM (Mediatech) with highglucose, L-glutamine and 10% fetal bovine serum and maintained at 37′Cwith 5% CO₂. Tumor cells were authenticated through ATCC CellAuthentication testing services by way of PCR/short tandem repeat (STR)profiling. Cells were routinely tested for mycoplasma using theUniversal Mycoplasma Detection Kit (ATCC, 30-1012K), and found to befree of contamination.

Twenty-nine female, SCID mice between the ages of 5-8 weeks of age wereused in these studies. All mouse feeding, husbandry and veterinary wasmanaged by the EMSS under IACUC approved guidelines and protocols. Micewere caged in groups of four or less, and fed and watered ad libitum.Prior to injection, mice were shaved to ascertain a suitable site forthe tumor xenografts. Tumor cells (10×10⁶ cells in Matrigel™; BDBioscience) were injected subcutaneously on the left rear flank of eachmouse. Tumor burden evaluations were made twice weekly using electroniccalipers to determine tumor size ((a2×b2)/2). When tumors reached a meanvolume of 500-700 mm³ the mice were randomized to one of 6 groups: 3groups of mice for tumor O₇ measurements and 3 groups of mice for tumorgrowth measurements.

Tumor pO₂ measurements were performed in 9 of the mice. NVX-108 doses of0.3 (2 mice), 0.45 (3 mice) and 0.6 (4 mice) mL/kg (2% w/vol emulsion)were administered. Tumor growth rate was studied in the other 20 mice.These mice were designated to the following 3 treatment groups: Group 1:No treatment (4 mice), Group 2: breathing carbogen while being treatedwith a 12 Gy radiation dose (8 mice), Group 3: Treated with NVX-108 (0.6cc per kg, 2% w/vol dodecafluoropentane (DDFP) administered IV over 30minutes with radiation at end of infusion) and breathing carbogen whilebeing treated with a 12 Gy radiation dose (8 mice).

Each mouse received ketamine (20 mg/kg IP) with xylazine (5 mg/kg IP)for immobilization and was fitted with a tail-vein catheter for dosingby tail vein injection (TVI). The tail-vein catheterization was achievedby using a fabricated 27-gauge needle catheter retrofitted to PE 50tubing. The catheter was firmly affixed to each animal's tail using 3-0suture thread and specialized adhesive tape on both sides of the tail.Following catheterization, the mice were placed within a custom-builtgas chamber introducing carbogen (95%02, 5% CO₂) which was ventilated atthe 10-minute mark. They were held in custom restraints in order to bepositioned under a lead shield isolating the flank tumor xenograft forradiation fractionation. All mice underwent a single fractionation of 12Gy to the tumor within a custom-built gas chamber introducing carbogen(95% O₂, 5% CO₂) which was ventilated at the 10-minute mark. Thecalculation for the XRT dose duration was based on “prescribed dose/doserate”=(1200 cGy)/(87.9 cGy/min) amounts to a 13.65 minute (13 m39 s)single fraction delivered to the tumor. For group #2, 200 μL sterilesaline was introduced via tail vein injection (TVI) (Time 0:00) andserved as the sham injection commencing 10 minutes prior to carbogenbreathing and 16.35 minutes (16 m21 s) before irradiation. For group #3,200 μL of NVX-108 was injected by way of TVI (Time 0:00) 10 minutesbefore carbogen breathing and 16.35 minutes before irradiation. Tailvein injections were performed with a multi-syringe pump forsimultaneous administration to each group. Simultaneous injections ofNVX-108 and saline initiated the study at time 0:00 minutes. This wasfollowed by carbogen breathing at 10 minutes and then radiation at 16.35minutes.

Once radiation was completed, the mice were allowed to recover and havefood and water ad libitum. Tumor size bi-dimensional measurements wereperformed twice per week. When tumor size was greater than 2000 mm3,mice were sacrificed.

Tumor oxygenation and blood flow levels were monitored using OxyLab(Oxford Optronics, Oxford, UK) triple parameter E-series fiber-opticprobes stereotactically inserted into all tumors. Oxygen partialpressure signals from these monitors were recorded in real-time using amulti-channel data acquisition system (PowerLab 8SP, ADInstruments,Australia) running under Chart™ for Windows™ (Ver.5.02. ADInstruments,Australia). Anesthetized mice (Isoflurane®), 100% O₂) were restrained ona custom immobilization platform to prevent movement and retrofittedwith a heating pad to sustain body core temperature. Precaution wastaken to prevent any movement of the hypoxia probes and eliminateinterference from external light sources to prevent probe artifact.Tumors were penetrated using a 19 gauge needle to a depth of 2-4 mm andmicroprobes (OD ˜450 μm) were fed through the needle into the tumorxenografts and fixed in position using stereotactic methods. Microprobeswere carefully marked with gradations in order to reach the same depthin all tumors. Once the probes were stabilized and immobilized theoutput signals were monitored (5-10 min) until a stable baseline wasobserved. Real-time measurements were taken for 10 minutes at baselineon carbogen and following a 200 μL IV injection via tail vein of dosesof 0.3, 0.45 or 0.6 cc/kg NVX-108 (NuvOx Pharma Tucson, Ariz.) whileanimals continued to breathe carbogen.

Example 3 Treatment of Glioblastoma Multiforme (GBM)

A patient with GBM undergoes surgery. The post-surgery gadoliniumenhanced MRI scan shows residual enhancing tumor. The patient is treatedwith 30 fractions of radiotherapy of 2 Gray (Gy) each over 6 weeks for atotal of 60 Gy with oral administration of temozolamide day, at a doseof 75 mg per square meter per day given 7 days per week from the firstday of radiotherapy until the last day of radiotherapy. The patientreceives an intravenous PICC line. DDFPc is administered at a dose of0.05 cc/kg (2% w/vol) as IV infusion over 30 minutes with infusioncommencing about 30 minutes prior to initiation of each radiotherapysession. Magnetic resonance TOLD scan is performed to show reversal oftumor hypoxia.

Follow-up MRI scans performed with intravenous gadolinium contrast showdecrease in tumor compared to patients treated without DDFPe.

Baseline post-operative MRI scan is shown hereinbelow to left. Whitearrow designates residual enhancing tumor seen in medial left temporallobe. Scan to right is 4 weeks after completion of chemo-irradiation andtreatment with DDFPe. White arrow designates residual enhancing tumor.Enhancing tumor has decreased by about 80%. Patient is now alive anddoing well more than 6-months after completion of therapy.

Example 4 Treatment of Glioblastoma Multiforme (GBM)

Another patient with glioblastoma undergoes surgery and has residualtumor visualized on contrast enhanced MRI. The patient is treated as inExample 1 (above) except using a dose of 0.1 cc/kg of DDFPe. The patienttolerates the treatment well. The next patient, presently beingconsented will be treated with a dose of 0.17 cc/kg of DDFPe during eachfraction of chemo-irradiation.

Prophetic Example 1

A patient with non-small cell lung cancer is treated with thoracicradiotherapy and concomitant chemotherapy as described by the protocolby Belani, et al. “Sequential chemotherapy consisted of two 3-weekcycles of paclitaxel 200 mg/m² administered over 3 hours, immediatelyfollowed by carboplatin at an area under the plasma concentration timecurve (AUC)=6 mg/mL min as an intravenous infusion over 30 minute.Thoracic radiotherapy is initiated on day 42 and consists of 1.8 Gydaily, five times per week (45.0 Gy target dose in 5 weeks to theinitial field), followed by a total of 18.0 Gy fractions delivered at2.0 Gy fractions daily to the initial tumor volume with reduced fields(total dose, 63.0 Gy in 34 fractions over 7 weeks), but includingenlarged lymph nodes ≥2.0 cm·3″ DDFPe is administered IV as an infusionover 15 minutes, commencing 30 minutes before the initiation orradiotherapy (RT), for each fraction of RT. Patients treated with DDFPeshow improved response to the regimen.

Prophetic Example 2

A patient with Stage I non-small cell lung cancer is treated with ahypo-fractionated radiotherapy schedule with three fractions of 15 Gy toa total of 45 Gy during 1 week. This represents a biological equivalentdose (BED) of 112.5 Gy. Between 30 to 60 minutes prior to each radiationdose, the patient is administered a bolus IV dose of 0.17 cc/kg NVX-108(2% w/vol DDFPe). Follow-up shows greater eradication of the treatedtumor than would be observed without DDFPe.

Prophetic Example 3

A female patient with cervical carcinoma is treated with combinedradiation therapy and chemotherapy+NVX-108. Radiation dosage is 45 Gray(Gy) in 20 fractions followed by low dose-rate intracavitary applicationof 30 Gy to the cervical region. Chemotherapy consists of intravenouscisplatin 40 mg/m2 every week for up to 6 weekly cycles. The patient isadministered a bolus IV dose of 0.2 cc/kg NVX-108 (2% w/vol DDFPe) 60minutes prior to each dose of radiation. Follow-up shows completeresponse to treatment.

Prophetic Example 4

A patient with squamous cell carcinoma of the head and neck is treatedwith 0.50 units/kg (20 units/m2) of bleomycin intravenously twiceweekly. During each administration of bleomycin the patient isadministered 0.2 cc/kg of 2% w/vol perfluorohexane emulsion whilebreathing carbogen (98% O₂/2% CO₂). The increased oxygen levels attainedin the tumor tissue increase the activity of the bleomycin and animproved response is attained.

Prophetic Example 5

An adult patient with germ cell ovarian cancer is treated withdactinomycin 500 mcg/day for 5 day: every 4 weeks. Each vial ofdactinomycin contains 0.5 mg (500 meg) of dactinomycin and 20 mg ofmannitol and is administered IV to the patient. DDFPe (0.2 cc/kg, 2%w/vol DDFP) is infused as an IV bolus concomitantly with eachadministration of dactinomycin. The patient breathes carbogen for 30minutes during and after the infusion. Increased levels of oxygen in thetumor tissue are attained, enhancing the activity of the drug.

Prophetic Example 6

An adult patient with rhabdomyosarcoma is treated with IV Vincristine ata dose of 1.4 mg/m2. Concomitantly the patient is administered 0.1 cc kgof DDFPc while breathing room air. Despite breathing room air, increasedoxygen levels am still attained in the tumor tissue resulting inincreased activity of the drug.

Prophetic Example 7

A patient with multiple myeloma is treated with BiCNU® (carmustine forinjection), a nitrosourea (1,3-bis(2-chloroethyl)-1-nitrosourea) incombination with prednisone. The dose of BiCNU administered to thispreviously untreated patient is 200 mg/m2 intravenously every 6 weeks.This is divided into daily injections of 100 mg/m2 on 2 successive days.DDFPe is administered as an IV bolus (dose=0.2 cc/kg, 2% w/vol DDFP)during each dose of BiCNU while the patient breathes supplemental oxygenfor 60 minutes. A repeat course of BiCNU is again administered once thecirculating blood elements have returned to acceptable levels (plateletsabove 100,000/mm3, leukocytes above 4,000/mm3), in 6 weeks, and againDDFPe is administered concomitantly with BiCNU.

Prophetic Example 8

A patient with prostate cancer is treated with combination external beamradiotherapy and high temporary seed implant high dose ratebrachytherapy. About 3 weeks after receiving 45Gy of external beamradiotherapy for prostate cancer temporary seed implant with high doserate brachytherapy is performed. The patient is brought to the operatingroom and anesthesia is induced. A transrectal ultrasound probe isintroduced into the rectum and the probe is then secured into a floormounted stepping device. A needle guide/perineal template is attached tothe stepping unit and pushed up against the perineal skin. Twenty metalneedles are placed through the template, pushed through the perineum,and advanced to the mid-prostate gland. The needles are replaced withplastic catheters. Upon recovery, the patient is brought to theRadiation Oncology department. CT scanning is performed to confirm theaccuracy of the catheter placements. This computer-controlled high doseradiotherapy unit contains a source drive mechanism that moves theradioactive Iridium wire through the interstitial catheters sequentiallyin accordance with the loading pattern determined by the dosimetry plan.It takes about 10-15 minutes for the high dose rate brachytherapyprocedure to be performed wherein the iridium wire is advanced into eachof the interstitial catheters. This is repeated one more time and thepatient is transferred to the hospital room for 6-hours, and the processis repeated again, for two more high dose rate brachytherapyadministrations, e.g. the iridium wire is advanced into each catheter atotal of four times, twice in the morning and twice in the afternoon,each session taking a total of about 30-minutes. During each treatmentsession the patient is administered a bolus dose of 0.2 cc/kg DDFPc over30 minutes while breathing carbogen.http://prostate-cancer.org/temporary-seed-implant-with-high-dose-rate-brachytherapy/

Prophetic Example 9

A pediatric patient with Stage IV Wilms tumor is treated withdactinomycin, doxorubicin, cyclophosphamide and vincristine for 65weeks. Doses of the drugs are as follows: dactinomycin (15 mcg/kg/d[IV]), vincristine (1.5 mg/m 2 wk [IV)), Adriamycin (doxorubicin 20 mg/m2/d [IV]), and cyclophosphamide (10 mg/kg/d [IV]). Dactinomycin coursesare given postoperatively and at 13, 26, 39, 52, and 65 weeks.Vincristine is given on days 1 and 8 of each Adriamycin course.Adriamycin is given for three daily doses at 6, 19, 32, 45, and 58weeks. Cyclophosphamide is given for three daily doses during eachAdriamycin and each dactinomycin course except the postoperativedactinomycin course. During each administration of dactinomycin andvincristine a dose of 0.2 cc/kg of DDFPe is administered while thepatient breathes supplemental oxygen. * D'angio, Giulio J., et al.“Treatment of Wilms' tumor. Results of the third national Wilms' tumorstudy.” Cancer 64.2 (1989): 349-360.

Prophetic Example 10

A patient with unrespectable hepatocellular carcinoma is under treatmentwith sorafenib. The patient is receiving 400 mg per day of oralsorafenib (2×200 mg). In a single setting the patient is also treatedwith TheraSphere which consists of insoluble glass microspheres whereyttrium-90 is bound within the spheres. The hepatic artery iscatheterized and the tumor vascular bed is embolized with TheraSpheredelivering a target dose of TheraSphere of 100 Gy by injection throughthe hepatic artery. A dose of 0.1 cc per kg of DDFPe is mixed withoxygen and is also infused into the hepatic artery during theembolization procedure.

Prophetic Example 11

Xenograft tumors were generated in mice with cell lines of UTSCC33 (oralcarcinoma), FADUDD (a subline of FaDu, an undifferentiatedhypopharyngeal carcinoma) and SiHa uterine cervix carcinoma.HPV-positive, (obtained from the American Type Culture Collection) aspreviously described. See, Toustrup, Kasper, et al. “Development of ahypoxia gene expression classifier with predictive impact for hypoxicmodification of radiotherapy in head and neck cancer.” Cancer research71.17 (2011): 5923-5931 (hereinafter “Toustrup”).

Mice bearing each kind of tumor were randomly assigned to two groups,DDFPe treatment and control (injected with same volume of saline). DDFPetreatment comprised administration of 0.3 cc/kg of DDFPe IV as boluseach day for 14 days. After the 14th day the mice were sacrificed andthe tumors assayed for expression of hypoxia related genes. See,Toustrup RNA from fresh-frozen tissue was extracted by using RNeasy-kit(Qiagen) according to the manufacturer's instructions. cDNA wasgenerated by using the High Capacity cDNA Archive kit (AppliedBiosystems; ABI) and gene expression was quantified by using qPCR. cDNAbased on FFPE samples was preamplified according to the manufacturer'sdetails (TaqMan PreAmp, ABI) before real time qPCR. To detecttranscripts of interest, TaqMan Gene Expression assay (ABI) was used forall potential classifier and reference genes. Genes of interest (knownto be upregulated in hypoxic tumors and associated with tumors mostlikely to progress) included the following: ADM (stress response), ALDOA(glucose metabolism), ANKRD37 (protein-protein interactions), BNIP3(apoptosis), BNIP3L (apoptosis), C3orf28 (unknown), EGNL3 (regulation ofHIF-1 activity), KCTD11 (apoptosis), LOX (extracellular matrixmetabolism), NDRG1 (stress response), P4HA1 (extracellular-matrixmetabolism). P4HA2 (extracellular matrix metabolism), PDK1 (energymetabolism), PFKFB3 (glucose metabolism) and SLC2A1 (glucosemetabolism). Assay of gene expression in the tumor xenografts from theanimals treated with DDFPe showed significantly lower expression of thehypoxia related genes than in tumor tissue specimen derived from theanimals treated with saline control injections.

Prophetic Example 12

The following prophetic example is meant to show how administration ofDDFPe can downregulate expression of genes that are over expressed inhypoxic tumor tissue and upregulate expression of genes that areexpressed in normoxic tissue (i.e. normalize gene expression). Fischer344 rats (F344/Ncr; National Cancer Institute, Frederick, Md.) were usedto generate 9 L glioma tumor models. Pieces of 9 L glioma were tied intothe epigastric artery/epigastric vein pair as previously described. Theanimals received daily IV injections of either 0.45 cc/kg DDFPe orsaline until the tumors weighed approximately 1.5-g at which time theanimals were euthanized, the tumors removed and flash frozen. Geneexpression in the tumors was assayed similarly to that described above.Up-regulated genes seen in the control group included BCL2/adenovirusEIB 19 kDa-interacting protein 3, homo oxygenase (decycling) 1,activating transcription factor 3, heat shock protein (HSP27), N-mycdownstream regulated gene 1, carbonic anhydrase 9 and others. Genes thatwere downregulated in the control group included Ly6-C antigen, solutecarrier family 44 (member2), sterile alpha motif domain containing9-like, DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 and CD3 molecule deltapolypeptide and others. Comparison of gene expression from 9-L gliomatissues from the animals treated with DDFPe showed significant decreasein expression of the genes that were upregulated in the control animalsand significant increase in the genes that were downregulated in thecontrol animals; i.e. there was normalization of gene expression in thetumors from animals treated with DDFPe. See, Marotta, Diane, et al. “Invivo profiling of hypoxic gene expression in gliomas using the hypoxiamarker EF5 and laser-capture microdissection.” Cancer research 71.3(2011): 779-789.

Prophetic Example 13

A 30% sucrose solution was prepared as described in example 1. In asecond vessel a suspension of a mixture of phospholipids with thefollowing composition. DPPC and DPPE-PEG 5 k in a mole ratio of 92% DPPCand 8 mole percent DPPE-PEG was prepared by warming them in water toabove the phase transition temperature of the all the lipids. Once thelipids were dispersed the suspension was cooled to 4 C and stirred in ajacketed vessel. Pre-cooled (4° C.) DDFP was added to the stirredphospholipid suspension at weight ratio of 7 to 1, and allowed to stiruntil a uniformly milky suspension was achieved. This suspension washomogenized under high pressure in an Avestin model C50 homogenizer forup to 18 minutes keeping the temperature below 7° C. The emulsion wastransferred via the homogenizer under low pressure to a vesselcontaining 30% sucrose solution in water; the resulting solution isstirred for up to 20 minutes, and then transferred through thehomogenizer under low pressure to a second vessel. This solution wasthen transferred through a 0.2 micron filter into a third vessel. Theproduct was dispensed to vials, which were capped and crimped. Theseoperations were carried out at <8° C. in cold jacketed vessels due tothe volatility of the active ingredient (DDFP). Compensation for lossesduring processing are accounted for by the use of an overage of theactive component. Product fill volume was also tightly controlled toproduce vials to meet release and shelf-life specifications.

Prophetic Example 14

A 30% sucrose solution was prepared as described in example 1. In asecond vessel a suspension of a mixture of phospholipids with thefollowing composition, DPPC and DPPE-PEG 5 k was prepared by warmingthem in water to above the phase transition temperature of the all thelipids. Once the lipids are dispersed the suspension was cooled to 4° C.and stirred in a jacketed vessel. Pre-cooled (4° C.) perfluorohexane wasadded to the stirred phospholipid suspension at weight ratio of 7 to 1,and allowed to stir until a uniformly milky suspension was achieved.This suspension was homogenized under high pressure in an Avestin modelC50 homogenizer for up to 18 minutes keeping the temperature below 7°C.). The emulsion was transferred via the homogenizer under low pressureto a vessel containing 30% sucrose solution in water, the resultingsolution was stirred for up to 20 minutes, and then transferred throughthe homogenizer under low pressure to a second vessel. This solution wasthen transferred through a 0.2 micron filter into a third vessel. Theproduct was dispensed to vials, which were capped and crimped. Theseoperations were carried out at <8° C. in cold jacketed vessels due tothe volatility of the active ingredient (perfluorohexane). Compensationfor losses during processing were accounted for by the use of an overageof the active component. Product fill volume was also tightly controlledto produce vials to meet release and shelf-life specifications.

Prophetic Example 15

A 30% sucrose solution was prepared as described in example 1. In asecond vessel a suspension of a mixture of phospholipids with thefollowing composition, DPPC, cholesterol and DPPE-PEG 5 k was preparedby warming them in water to above the phase transition temperature ofthe all the lipids. Once the lipids were dispersed the suspension wascooled to 4° C. and stirred in a jacketed vessel. Pre-cooled (4° C.)perfluoroheptane was added to the stirred phospholipid suspension atweight ratio of 7 to 1, and allowed to stir until a uniformly milkysuspension was achieved. This suspension was homogenized under highpressure in an Avestin model C50 homogenizer for up to 18 minuteskeeping the temperature below 7° C. The emulsion was transferred via thehomogenizer under low pressure to a vessel containing 30% sucrosesolution in water; the resulting solution was stirred for up to 20minutes, and then transferred through the homogenizer under low pressureto a second vessel. This solution was then transferred through a 0.2micron filter into a third vessel. The product was dispensed to vials,which were capped and crimped. These operations were carried out at <8°C. in cold jacketed vessels due to the volatility of the activeingredient (perfluoroheptane). Compensation for losses during processingwere accounted for by the use of an overage of the active component.Product fill volume was also tightly controlled to produce vials to meetrelease and shelf-life specifications.

Prophetic Example 16

A 30% sucrose solution was prepared as described in example 1. In asecond vessel a suspension of a mixture of phospholipids with thefollowing composition, DPPC, phosphatidic acid (DPPA) and DPPE-PEG 5 kwas prepared by warming them in water to above the phase transitiontemperature of the all the lipids. Once the lipids were dispersed thesuspension was cooled to 4° C. and stirred in a jacketed vessel.Pre-cooled (4° C.) perfluorooctane was added to the stirred phospholipidsuspension at weight ratio of 7 to 1, and allowed to stir until auniformly milky suspension was achieved. This suspension was homogenizedunder high pressure in an Avestin model C50 homogenizer for up to 18minutes keeping the temperature below 7° C. The emulsion was transferredvia the homogenizer under low pressure to a vessel containing 30%sucrose solution in water; the resulting solution is stirred for up to20 minutes, and then transferred through the homogenizer under lowpressure to a second vessel. This solution was then transferred througha 0.2 micron filter into a third vessel. The product was dispensed tovials, which were capped and crimped. These operations are carried outat <8° C. in cold jacketed vessels due to the volatility of the activeingredient (perfluorooctane). Compensation for losses during processingwere accounted for by the use of an overage of the active component.Product fill volume was also tightly controlled to produce vials to meetrelease and shelf-life specifications.

Prophetic Example 17

A suspension of a mixture of phospholipids with the followingcomposition, dioleoylphosphatidylcholine (DOPC), cholesterol anddioleoylphosphatidylethanolamine-PEG-5,000 was prepared by warming themin water to above the phase transition temperature of the all thelipids. The resulting suspension of lipids was suspended in a mixture ofpropylene glycol/glycerol to achieve 80:10:10 weight percent phosphatebuffered saline:propylene glycol:glycerol. Once the lipids weredispersed the suspension was cooled to 4 C and stirred in a jacketedvessel. Pre-cooled (4° C.) perfluorohexane was added to the stirredphospholipid suspension at weight ratio of 7 to 1, and allowed to stiruntil a uniformly milky suspension was achieved. This suspension washomogenized under high pressure in an Avestin model C50 homogenizer forup to 18 minutes keeping the temperature below 7° C. The emulsion wastransferred via the homogenizer under low pressure to a vesselcontaining 30% sucrose solution in water; the resulting solution isstirred for up to 20 minutes, and then transferred through thehomogenizer under low pressure to a second vessel. This solution wasthen transferred through a 0.2 micron filter into a third vessel. Theproduct was dispensed to vials, which were capped and crimped. Theseoperations are carried out at <8° C. in cold jacketed vessels due to thevolatility of the active ingredient (perfluorooctane). Compensation forlosses during processing were accounted for by the use of an overage ofthe active component. Product fill volume was also tightly controlled toproduce vials to meet release and shelf-life specifications.

While the preferred embodiments of the present invention have beenillustrated in detail, it should be apparent that modifications andadaptations to those embodiments may occur to one skilled in the artwithout departing from the scope of the present invention as set forthherein.

1-25. (canceled)
 26. A method of treating solid hypoxic tumors bymulti-fraction radiotherapy, comprising administering to a subjectsuffering from a solid hypoxic tumor, concomitantly or up to 120 minutesprior to each fraction of radiation therapy, a water emulsion ofperfluoropentane to the mammal, wherein the perfluoropentane emulsioncomprises a viscosity modifier, a fluorosurfactant, and about 1% to 10%w/vol of perfluoropentane, and the solid hypoxic tumor is selected frompancreatic cancer, non-small cell lung cancer, cervical carcinoma,squamous cell carcinoma, ovarian cancer, sarcoma, multiple myeloma,prostate cancer, rectal cancer, Wilms tumor, head and neck cancer, oralcarcinoma and uterine carcinoma.
 27. The method of claim 26, wherein thesubject is concomitantly administered a chemotherapeutic agent.
 28. Themethod of claim 26, wherein the subject is concomitantly administered animmunosensitizing agent.
 29. The method of claim 26, wherein the solidhypoxic tumor is pancreatic cancer.
 30. The method of claim 26, whereinthe solid hypoxic tumor is non-small cell lung cancer.
 31. The method ofclaim 26, wherein the perfluoropentane emulsion comprises about 1% to 5%w/vol of perfluoropentane.
 32. The method of claim 31, wherein theperfluoropentane emulsion is administered at a dosage in the range fromabout 0.05 cc/kg to about 0.3 cc/kg.
 33. The method of claim 32, whereinadministration of the perfluoropentane emulsion is intravenous infusionfor up to 30 minutes.
 34. The method of claim 32, wherein administrationof the perfluoropentane emulsion is intravenous infusion as a singlebolus.
 35. The method of claim 26, wherein the fluorosurfactant is PEGTelomer B.
 36. The method of claim 35, wherein the fluorosurfactant isPEG Telomer B is present at about 3 mg/mL.
 37. The method of claim 26,wherein the viscosity modifier comprises sucrose.
 38. The method ofclaim 37, wherein the sucrose is present at about 300 mg/mL.
 39. Themethod of claim 26, wherein the perfluoropentane emulsion isco-administered with an anticancer agent.
 40. The method of claim 39,wherein the anticancer agent is an antibody.
 41. The method of claim 26,wherein the subject is concomitantly administered a chemotherapeutic andan immunosensitizing agent.